Activation-induced modification in the CD3 complex of the gammadelta T cell receptor

Abstract

The T cell antigen receptor complexes expressed on alphabeta and gammadelta T cells differ not only in their respective clonotypic heterodimers but also in the subunit composition of their CD3 complexes. The gammadelta T cell receptors (TCRs) expressed on ex vivo gammadelta T cells lack CD3delta, whereas alphabeta TCRs contain CD3delta. While this result correlates with the phenotype of CD3delta(-/-) mice, in which gammadelta T cell development is unaffected, it is inconsistent with the results of previous studies reporting that CD3delta is a component of the gammadelta TCR. Since earlier studies examined the subunit composition of gammadelta TCRs expressed on activated and expanded peripheral gammadelta T cells or gammadelta TCR(+) intestinal intraepithelial lymphocytes, we hypothesized that activation and expansion may lead to changes in the CD3 subunit composition of the gammadelta TCR. Here, we report that activation and expansion do in fact result in the inclusion of a protein, comparable in mass and mobility to CD3delta, in the gammadelta TCR. Further analyses revealed that this protein is not CD3delta, but instead is a differentially glycosylated form of CD3gamma. These results provide further evidence for a major difference in the subunit composition of alphabeta- and gammadelta TCR complexes and raise the possibility that modification of CD3gamma may have important functional consequences in activated gammadelta T cells.

Figure 1.

Effect of in vitro activation and expansion on the subunit composition of αβ- and γδ TCR complexes. Ex vivo αβ and γδ T cells were purified from the lymph nodes of B6 and γδ TCR Tg mice, respectively. Stimulated αβ and γδ T cells, from B6 and γδ TCR Tg mice, respectively, were generated as described in Materials and Methods. Surface proteins were labeled with biotin and αβ and γδ TCR complexes were immunoprecipitated using anti-TCR mAbs (H57–597 and UC7–13D5, respectively) or anti-TCR-ζ mAb (H146). Immunoprecipitated proteins were resolved by nonreducing/reducing 2-D SDS-PAGE. ABC-HRP and chemiluminescence were used to visualize surface biotinylated proteins. The positions of the CD3 subunits and the unknown 26-kD subunit (?) are marked.

Figure 2.

Biochemical analysis of CD3 complexes expressed by stimulated γδ T cells. (A) Lysates from 20 × 10⁶ ex vivo αβ and γδ T cells and stimulated γδ T cells were immunoprecipitated with anti–TCR-ζ mAb (H146). Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-CD3δ serum. The blot was subsequently stripped and probed with anti-CD3ɛ to assess the efficiency of immunoprecipitation. (B) Total cellular CD3δ protein levels in ex vivo and in stimulated αβ and γδ T cells. Extracts were made from 10 × 10⁶ cells and 0.3 × 10⁶ cell equivalents were then analyzed by immunoblotting with anti-CD3δ serum. (C) Lysates from 25 × 10⁶ stimulated V_γ1-J_γ4-C_γ4⁺ γδ T cells and B6 thymocytes were immunoprecipitated with anti-TCR mAbs (UC7–13D5 and H57–597, respectively). Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-CD3δ serum. The blot was subsequently stripped and probed with anti-CD3ɛ to assess the efficiency of immunoprecipitation. (D) 20 × 10⁶ stimulated γδ T cells from nontransgenic CD3δ^−/− mice were surface biotinylated, lysed, and incubated with anti-CD3ɛ mAb (145–2C11). Immunoprecipitated proteins were resolved by nonreducing/reducing 2-D SDS-PAGE. Biotinylated TCR subunits were detected with ABC-HRP and chemiluminescence. The positions of the CD3 subunits and the unknown 26-kD subunit (?) are marked.

Figure 3.

Modification of the CD3γ subunit after in vitro activation and expansion. (A) 30 × 10⁶ ex vivo γδ (γδ TCR Tg [Tg] CD3δ^+/− mice) and stimulated γδ T cells (Tg CD3δ^+/− and Tg CD3δ^−/− mice) were surface biotinylated, lysed, and incubated with anti-CD3ɛ mAb (145–2C11). Immunoprecipitated proteins were either treated with PNGase F or left untreated and resolved by reducing SDS-PAGE. ABC-HRP and chemiluminescence were used to visualize surface biotinylated proteins. The positions of the CD3 subunits and the unknown 26-kD subunit (?) are marked. (B) Lysates from 30 × 10⁶ ex vivo γδ (Tg CD3δ^+/− mice) and stimulated γδ T cells (Tg CD3δ^+/− and Tg CD3δ^−/− mice) were immunoprecipitated with anti-TCR-ζ mAb (H146) and then treated with PNGase F or left untreated. Digested and undigested TCR proteins were resolved by reducing SDS-PAGE and immunoblotted with anti-CD3γ serum. (C) Lysates from 30 × 10⁶ ex vivo αβ (B6 mice) and stimulated αβ T cells (B6 and CD3δ^−/− mice) were immunoprecipitated with anti-CD3ɛ (145–2C11) or anti-TCR-ζ mAb (H146). Immunoprecipitated proteins were resolved by reducing SDS-PAGE and immunoblotted with anti-CD3γ serum. (D) Stimulated αβ and γδ T cells from CD3δ²/⁻ mice were generated as described in Materials and Methods. Surface proteins on equivalent numbers of cells were labeled with biotin and TCR complexes were immunoprecipitated using the anti-CD3ɛ mAb (145–2C11). Immunoprecipitated proteins were resolved by nonreducing/reducing 2-D SDS-PAGE. ABC-HRP and chemiluminescence were used to visualize surface biotinylated proteins. The exposure time for each blot is identical. The positions of the CD3ɛ and the modified forms of CD3γ are marked.

Figure 4.

Detection of differentially glycosylated forms of CD3γ in iIELs. (A) Lysate from 30 × 10⁶ iIELs from B6 mice was subjected to serial immunoprecipitations with anti-γδTCR and then anti-αβTCR mAbs (UC7–13D5 and H57–597, respectively). Immunoprecipitated proteins were resolved by reducing SDS-PAGE and immunoblotted with anti-CD3δ serum. The blot was subsequently stripped and immunoblotted with anti-CD3γ serum. (B) Lysates from 20 × 10⁶ ex vivo iIELs from B6 mice were immunoprecipitated with anti-CD3ɛ (145–2C11). Immunoprecipitated proteins were resolved by reducing SDS-PAGE and immunoblotted with anti-CD3γ serum.

Figure 4.

Detection of differentially glycosylated forms of CD3γ in iIELs. (A) Lysate from 30 × 10⁶ iIELs from B6 mice was subjected to serial immunoprecipitations with anti-γδTCR and then anti-αβTCR mAbs (UC7–13D5 and H57–597, respectively). Immunoprecipitated proteins were resolved by reducing SDS-PAGE and immunoblotted with anti-CD3δ serum. The blot was subsequently stripped and immunoblotted with anti-CD3γ serum. (B) Lysates from 20 × 10⁶ ex vivo iIELs from B6 mice were immunoprecipitated with anti-CD3ɛ (145–2C11). Immunoprecipitated proteins were resolved by reducing SDS-PAGE and immunoblotted with anti-CD3γ serum.